We study the mechanism and regulation of eukaryotic protein synthesis focusing on the roles of GTPases and a family of stress-responsive protein kinases. In the first step of protein synthesis translation initiation factors promote the assembly of an 80S ribosome at the AUG codon of an mRNA. The factor eIF2 is a three-subunit complex that facilitates binding of the specific initiator methionyl-tRNA (Met-tRNA) to the small ribosomal subunit. The eIF2 binds GTP, and the initiator Met-tRNA to form a stable eIF2?GTP?Met-tRNA ternary complex. The gamma subunit of eIF2 contains the GTP-binding domain of the factor, and in collaboration with Stephen Burley at The Rockefeller University we obtained the crystal structure of eIF2gamma from the archaeon Methanococcus jannaschii. The eIF2gamma structure resembled the structure of bacterial elongation factor EF-Tu. A mutation in the putative tRNA-binding pocket of eIF2gamma impaired yeast cell growth and was suppressed by overexpression of initiator tRNA. In addition, mutation of conserved surface residues adjacent to the tRNA binding pocket in yeast eIF2gamma caused slow growth that was suppressed by overexpression of eIF2alpha. Biochemical analysis of the corresponding mutations in archaeal eIF2gamma confirmed that the mutations disrupted the binding to eIF2alpha. Following AUG codon recognition eIF2gamma hydrolyzes its GTP triggering release of the factor from the ribosome. The factor eIF5B is a ribosome-dependent GTPase that promotes subunit joining in the final step of translation initiation. The eIF5B is an ortholog of the prokaryotic translation factor IF2. In collaboration with Stephen Burley we determined the X-ray structure of archaeal eIF5B. The chalice-shaped protein consists of four domains: the G domain and domains II and III form the cup of the chalice, a long alpha helix forms the stem, and domain IV is the base of the chalice. Yeast eIF5B contains a long N-terminal extension when compared to the archaeal factor. Deletion of this N-terminal region had no impact on yeast cell growth; however, deletion of the C-terminal ~25 residues of eIF5B slightly impaired yeast cell growth, and this growth defect was exacerbated by deletion of the N-terminal region of eIF5B. We propose that this growth defect reflects impaired ribosome binding because the C-terminal domain IV of eIF5B binds the factor eIF1A, which associates with the small ribosomal subunit. To assess the role of GTP-binding and hydrolysis by eIF5B, we mutated the Switch 1 (Sw1) and Switch 2 (Sw2) motifs in the factor's GTP-binding domain. Intragenic suppressors of the Sw1 mutant uncoupled eIF5B GTPase and translation stimulatory activities suggesting a regulatory rather than mechanical role for eIF5B GTP hydrolysis in translation initiation. We propose that a GTP-regulated switch governs the ribosome affinity of eIF5B. In the presence of GTP eIF5B binds the ribosome, and following GTP hydrolysis the factor is released. Mutation of a conserved glycine in Sw2 impaired eIF5B GTP-binding and hydrolysis activities and yeast cell growth. An intragenic suppressor of the Sw2 mutation mapped to Sw1 and restored GTP binding and hydrolysis; thus, providing evidence that the Sw1 and Sw2 elements in a G domain cooperate to promote GTP binding and hydrolysis. Four protein kinases PKR, GCN2, HRI and PERK regulate translation by phosphorylating serine-51 on the alpha subunit of eIF2, converting eIF2 from a substrate to a competitive inhibitor of its guanine nucleotide exchange factor eIF2B. The kinase PKR contributes to anti-viral defense in mammalian cells, and we established a heterologous system in yeast to study PKR. High-level expression of PKR inhibits yeast cell growth, and co-expression of the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR, alleviates PKR toxicity in yeast. Twelve single amino acid changes were identified in the PKR kinase domain that restored PKR toxicity in yeast co-expressing the K3L protein. We propose that these mutations, located in the PKR kinase domain, lower the affinity of PKR for its pseudosubstrate without severely impairing substrate binding and phosphorylation. N- and C-terminal truncation analyses revealed that residues 1-180 of eIF2alpha represent the minimal substrate for efficient phosphorylation of serine-51 by PKR or GCN2. Mutations were isolated throughout the eIF2alpha N-terminus that impaired phosphorylation of serine-51 by GCN2 and PKR both in vivo and in vitro. Strikingly, substitution of alanine for aspartic acid-83, 32 residues from the site of phosphorylation, completely blocked phosphorylation. We propose that the eIF2alpha kinases recognize their substrate utilizing residues both nearby and remote from the phosphorylation site. A second set of mutations in the eIF2alpha N-terminus blocked translational regulation, and eIF2B binding, but not serine-51 phosphorylation indicating that the eIF2alpha kinases and eIF2B interact with overlapping surfaces on eIF2alpha.